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TECHNICAL STUDY
Mycoplasma removal
By Laurelle Sciola, Product Manager and David Yavorsky, R&D, Millipore Corporation
C
ell culture media is available in many different
types and variations designed to suit the spe-
cific needs of cell lines and genetically-modi-
fied variants. In addition, researchers have developed
an even wider variety of formulations – many of which
are chemically derived – to meet their specific culture
requirements and to mitigate the risk of contamina-
tion. Growth media remains an evolving technology
where change is driven by the increasing number of
production cell lines, as well as concerns over product
safety and purity.
Of key importance to ensuring sterility of the cell
culture batch and subsequent purification processing
is careful preparation of cell culture media. Cell culture
media must be sterilized before being delivered to an
aseptic bioreactor or fermenter to prevent the growth of
adventitious bacteria or other organisms. Mycoplasma
has become widely recognized as the smallest bacteri-
um thriving in cell culture and cannot be retained by
0.2µm sterilizing-grade filters.
Mycoplasma is the name given to microorganisms
in the class Mollicutes. These microorganisms lack a cell
wall and are bound together by a trilaminar plasma
membrane. Due to their small size and deformability,
mycoplasma can penetrate a 0.2µm rated filter.
Frequently, methods for media preparations are being
revised to replace 0.2µm sterilizing-grade filters with
0.1µm sterilizing-grade filters for sufficient log removal
of mycoplasma.
Media preparation
Media products are sold both as dry powdered
formulations and as pre-sterilized liquid solutions.
Small-scale operations (less than a few liters) may opt
for prepared liquid formulations that are ready-to-use.
Cost considerations, however, dictate that media
preparation for production-scale processes start with
dry ingredients dispersed into pharmaceutical-grade
water. Time, temperature and degree of agitation all
contribute to some degree of variability in the proper-
ties of the media, particularly with regard to the
amount and size of insoluble particulates – which, in
turn, impact filterability.
Pasteurization (short duration) is sometimes used
prior to sterile filtration as a means of reducing
pathogens other than bacteria, such as virus and pri-
Reprinted from NGP (US edition), Q1 2006 © GDS Publishing Ltd www.gdsinternational.com
ABOUT MILLIPORE CORPORATION
Millipore is a leading bioprocess and
bioscience products and services
company, organized into two divisions.
The Bioprocess Division offers solutions
that optimize development and
manufacturing of biologics. The Bioscience
Division provides high performance
products and application insights that
improve laboratory productivity. Millipore
has a deep understanding of its
customers’ research and manufacturing
process needs, and offers reliable and
innovative tools, technologies and
services. The Company employs
approximately 4850 people worldwide and
posted revenues of US$991 million in
2005. For more information about Millipore
visit www.millipore.com.
ons. Experience has shown that pasteurization process-
es typically cause no substantial changes to media
quality and filterability.
Media is typically mixed in bulk and then asepti-
cally transferred to the bioreactor. Pre-filtration is used
to remove the bulk of particular and colloidal contami-
nants from the media in order to reduce process biobur-
den and extend the service life of the filter train.
Pre-filters should be sized appropriately to handle
batch-to-batch media variability and ensure that the
sterile media fill in the bioreactor is completed suc-
cessfully and on time. Final sterilizing-grade filtration
should remove bacteria and mycoplasma to preserve
the aseptic barrier around the bioreactor.
Sterile filtration is generally a batch operation; pre-
pared media is transferred under pneumatic pressure
from a mix tank to the bioreactor or intermediate hold
tank though the sterilizing-grade filter(s). Sterile filters
are first steamed-in-place and often integrity-tested
prior to filtration. For smaller scale operations gamma-
sterilizable capsules, that are aseptically connected to
the process system, may be used.
Case study
Filters containing the Millipore Express SHR
sterilizing-grade polyethersulfone membrane offer
high levels of mycoplasma removal and high
throughput for cell culture media and other process
intermediates. Millipore Express SHR 0.1µm mem-
brane-containing filters are designed with an on-
board polyethersulfone membrane pre-filter that
protects the sterilizing-grade 0.1µm rated membrane
from premature plugging for enhanced throughput
in high fouling streams.
To illustrate the high throughput performance
of filters containing the Millipore Express SHR
membrane in cell culture media, these filters were
tested against other 0.1µm sterilizing-grade filters
with a range of media types; including CHO serum-
free media, NSO serum-free media, soy peptone-
based serum-free media and customer-specific
serum-free media. Competitive membranes tested
included Competitor A PES 0.2/0.1 and Competitor
B PVDF 0.2/0.1.
Experimental method
The 47mm membrane disks were wetted with 30
percent isopropyl alcohol and rinsed with water. Once
assembled in the stainless steel holder, they were in-
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TECHNICAL STUDY

tegrity tested to ensure proper membrane installation. Figure 1

To accomplish this, the holders were submerged in
water bath with filtrate open. At an air inlet pressure of
5psi, no visual air bubbles from the holders were no-
ticed. The membranes were then water flux tested, at a
fixed 10psi differential pressure for three minutes to en-
sure adequate wetting.
Feed solution temperatures were 21-26°C. Filter
pressures were fixed at 10psi differential and the feed
solution was stirred for test duration. A run consisted
of membrane samples tested simultaneously from a
single feed tank. For each membrane sample, the ac-
cumulated filtrate volume was weighed on a calibrat-
ed load cell every 4-5 seconds and the time/weight
recorded using a PC-based data acquisition system.
The tests were continued until 90 percent reduction in
initial membrane flux was reached or the feed solution
was exhausted. Time and volume data was analyzed
using a combined pore plugging model to estimate Figure 2
final throughput volumes at time infinity, or Vmax, for
each filter.

Results
The Millipore Express SHR membrane with Pre-
filter was very competitive in throughput for all media
evaluated. It demonstrated at least 60 percent
greater flux than its closest competitor. This flux ad-
vantage can contribute to media throughput particu-
larly for flux-driven filtrations (Figure 1).
For streams in which membrane plugging oc-
curred (measurable flow decay over the course of the
test), membrane performance is defined by through-
put, or filtrate volume per membrane area (in L/m2) at
set process times under a constant feed pressure
(10psid). Throughput was compared at a short time
interval to highlight those membranes with high flux
and also at a longer time interval to capture the mem-
brane’s plugging profile or performance limit.
Membrane ranking differed with the time intervals Figure 3
due to variation in membrane plugging profiles with
respect to time.
In two trials for non pre-filtered commercially
available CHO serum-free media, Millipore Express
SHR membranes with Pre-filter yielded greater than
2X throughput in both trials. At filtration times of more
than an hour, Vmax values were 9-10k L/m2 for
Millipore Express SHR membranes with Pre-filter.
These values were at least 5X greater versus
Competitor A PES 0.2/0.1 and Competitor B PVDF
0.2/0.1. The pre-filter layer used with the Millipore
Express SHR membrane provided adequate protec-
tion of the 0.1µm layer to result in extended flux-based
filtration times relative to other membranes (Figure 2).
Figure 3 shows the results of CHO media pre-fil-
tered with a Polysep II filter tested. Vmax values for
Polysep II filters were 800-900L/m2.

Reprinted from NGP (US edition), Q1 2006 © GDS Publishing Ltd www.gdsinternational.com
MILLIPORE ED 5/6/06 8:56 am Page 3

TECHNICAL STUDY

For final filtration, feed solution was exhausted

Figure 4 after 30 minutes in trial 1. Following Polysep II filtration,
a filter containing a Millipore Express SHR membrane
with Pre-filter provided sufficient protection of the 0.1µm
layer for flux-driven filtration and 2-3X Vmax improve-
ment over competitor filters.
Membrane requirements for final filtration of pre-
filtered customer supplied serum-free media are
shown in Figure 4. For pre-filtration, Polysep II filters
had Vmax values of ~600L/m2. A filter containing a
Millipore Express SHR membrane with Pre-filter and
Competitor A PES 0.2/0.1 were equivalent for area re-
quirements as values were within 10 percent Vmax
experimental error.
Figure 5 shows results of two trials for non pre-fil-
tered commercially available NSO serum-free media. At
a two-hour filtration time, a filter containing Millipore
Express SHR membrane with Pre-filter demonstrated 60
percent greater throughput than its closest competitor,
Competitor B PVDF 0.2/0.1. Longer filtration times widen
Figure 5
this performance gap, as Vmax is 2X greater for the filter
containing Millipore Express SHR membrane with Pre-
filter than Competitor B PVDF 0.2/0.1.
Filtration of non pre-filtered peptone-based
media is presented in Figure 6 for one trial. For all
membranes, except the filter containing Millipore
Express SHR membrane with Pre-filter, at least 80
percent of the final capacity or Vmax was reached
after 30 minutes filtration time. The resulting low flow
rates contributed very little to filtrate volume beyond
30 minutes. In this trial, the filter containing Millipore
Express SHR membrane with Pre-filter demonstrated
a 3-4X greater Vmax than competitive filters.
Additional testing of the filter containing
Millipore Express SHR membrane with Pre-filter and
Competitor A PES 0.2/0.1 was performed with pep-
tone-supplemental media at a lower concentration of
soy peptone (1g/L). Vmax of the filter containing
Millipore Express SHR membrane with Pre-filter ex-
Figure 6
ceeded Competitor A PES 0.2/0.1 by 6X.

Conclusions
Where sterilizing-grade performance and a high
degree of protection against mycoplasma contamina-
tion are required, the filter containing Millipore Express
SHR membrane offers high throughput values and low
area requirements for a majority of serum-free media
types tested with and without pre-filtration.
Filters with Millipore Express SHR membranes
are designed to withstand multiple sterilization cy-
cles, provide high flow rates, broad chemical com-
patibility (pH 1-14) and extended capacity for lower
filtration costs. The fast flowing, caustic stable filters
provide enhanced sterility assurance and high per-
formance with cell culture media, sera and other high
fouling biological process solutions.

Lit #: RP0019EN00